PROJECT SUMMARY Positive-stranded RNA viruses, including enteroviruses and flaviviruses, are responsible for severe disease manifestations worldwide. Enteroviruses, such as enterovirus 71 (EV71) and coxsackievirus B3 (CVB), enter the host via the fecal-oral route and, therefore, must initially cross the intestinal epithelium to cause severe disease, including acute flaccid paralysis and meningitis. Conversely, flaviviruses enter the host through the bite of an infect arthropod. Zika virus (ZIKV) and dengue virus (DENV) are transmitted by mosquitos in subtropical and tropical regions of the world. Importantly, several flaviviruses are known to cause severe neurological disease, including congenital Zika syndrome, which was first observed during a 2015 Brazilian outbreak. To cause neurological disease, the majority of viruses must cross the blood brain barrier. Thus, an understanding of cellular processes that regulate virus infection of barrier cells can facilitate the development of novel broad-range antiviral strategies and therapeutics. Interestingly, all positive-stranded RNA viruses require the manipulation of host membranes to concentrate viral and host factors at sites of viral replication. During infection enteroviruses and flaviviruses manipulate membranes of the endomembrane system, which connects the nuclear membrane to the extracellular space via vesicle trafficking between the endoplasmic reticulum (ER) and Golgi complex. Thus, I sought to better understand the shared cellular processes associated with the endomembrane system that are manipulated during virus infection. I directly compared ~50 host endomembrane factors for their ability to regulate enterovirus (EV71 and CVB) and flavivirus (ZIKV and DENV) infection. My preliminary results identified members of the reticulophagy regulator (RETREG) protein family and several soluble N-ethylmaleimide-sensitive associated receptor (SNARE) proteins, including vesicle associated membrane protein 7 (VAMP7). Interestingly, these proteins are all associated with autophagy, which is a cellular stress response pathway that is manipulated by enteroviruses and flaviviruses during infection. Thus, I hypothesize that select components of the endomembrane system regulate virus infection through facilitation of autophagic processes. To address the mechanisms of viral manipulation of the endomembrane system, I have developed plasmid-based reporters that will be used to monitor (1) ER and Golgi morphology and (2) induction of autophagy during infection and host factor depletion. These novel reporters will be used for long-term time-lapse imaging in a cellular model of the blood brain barrier. Furthermore, I will provide mechanistic insight into the role of RETREG proteins during enterovirus and flavivirus infection. Additionally, I will characterize enterovirus-induced autophagy using a blood brain barrier cell model and a highly relevant primary human intestinal epithelium model, that we have established in our laboratory. Information derived from our results will provide significant insight into the shared cellular processes manipulated by these viruses to efficiently replicate in cellular barriers.